CN105920773B - A kind of microbial inoculum and preparation method for triazine herbicide of degrading - Google Patents
A kind of microbial inoculum and preparation method for triazine herbicide of degrading Download PDFInfo
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Abstract
The present invention relates to a kind of microbial inoculums for triazine herbicide of degrading.The microbial inoculum is to embed bacillus albus using sodium alginate, polyvinyl alcohol crosslinked product as carrier（Leucobactersp.）JW-1 is made；The wet thallus content of bacillus albus category JW-1 bacterium is 30 g/L in microbial inoculum, polyvinyl alcohol content is 120 g/L, the content of sodium alginate is 20 g/L；Microbial inoculum is round granular, and surface is smooth, and elasticity is good, and mechanical strength is good, and transparent performance is good.Bacillus albus, which belongs to JW-1 bacterium, has good degradation effect to a variety of triazine herbicides, can quickly degrade containing methyl mercapto（-SCH3）And chloro（-Cl）Triazine herbicide.Free bacillus albus is easy to run off in the environment, adaptive capacity to environment is poor, therefore the bacterium bag is buried the fixed technical field of biological remediation that microbial inoculum is made and applies to triazine herbicide, has biggish biological prosthetic meaning and economic benefit.
Description
Technical field
The invention belongs to microorganism remediation organic contamination technical field, be related to a kind of triazine herbicide degradation bacterium preparation and
Preparation method and application.
Background technique
Triazine herbicide is one of current a kind of herbicide most popular in the world, and global triazines are removed within 2007
Careless agent sales volume accounts for herbicide market 5.3%.The kind listed at present has 11, as atrazine, Simanex, Garagard, propazine,
Prometryn, symetryne, ametryn, metribuzin etc..Triazine herbicide is widely used in the crop fields such as soybean, corn, sorghum, prevents
Except grass family and broadleaf weeds.Triazine herbicide has stronger adsorptivity because its chemical property is stablized in the soil, so
Half-life period in environment is up to even 1 year or more some months, triazine herbicide easily dirty soil, surface water and groundwater.It is some
Research shows that triazine herbicide can influence the endocrine system of mammality and amphibian animal, central nervous system and siberian crabapple
The development of system.Triazine herbicide is disabled by European Union at present, but it is still wide in Sino-America, Australia and Africa
General application.In face of the aggravation of environmental pollution, repairs pollution and become more and more important.
The pollution amelioration of triazine herbicide mainly has peripheral doses, chemical remediation and biological prosthetic.Peripheral doses and change
Learn repair because its reparation is not thorough, easily cause secondary pollution, rehabilitation cost is high the disadvantages of, practical application is simultaneously few.It is biological prosthetic
Technology has been increasingly becoming a kind of skill of the repairing environment pollution of mainstream because its is at low cost, repairs the advantages that thorough, without secondary pollution
Art means.
Summary of the invention
The problems such as to solve triazine herbicide residual contamination, it is an object of that present invention to provide one kind for triazines of degrading
The microbial inoculum and preparation method of herbicide.
Bacterium of the present invention for triazine herbicide of degrading is bacillus albus (Leucobacter sp.) JW-1,
Genbank accession number is KT439069 in NCBI, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number are CGMCC No：11754, preservation date：2015
November 27.
A kind of microbial inoculum for triazine herbicide of degrading is the packet using sodium alginate, polyvinyl alcohol crosslinked product as carrier
Bacillus albus (Leucobacter sp.) JW-1 is buried to be made；
The wet thallus content of bacillus albus (Leucobacter sp.) JW-1 is 30g/L in the microbial inoculum, polyvinyl alcohol contains
Amount is 120g/L, the content of sodium alginate is 20g/L；
Microbial inoculum is round granular, and surface is smooth, and elasticity is good, and mechanical strength is good, and transparent performance is good.
It prepares as follows for the operating procedure of the microbial inoculum for triazine herbicide of degrading：
(1) polyvinyl alcohol and sodium alginate are sequentially added in water, is stirred and evenly mixed, in 121 DEG C of pressure 101KPa, temperature items
Sterilize 20min under part, and room temperature is cooling, obtains mixed liquor；The content of polyvinyl alcohol is 120g/L, sodium alginate in the mixed liquor
For 20g/L；
(2) wet thallus of bacillus albus (Leucobacter sp.) JW-1 is added in mixed liquor, stirs and evenly mixs, obtains
Thallus embeds liquid；The content of the wet thallus of bacillus albus (Leucobacter sp.) JW-1 is 30g/L in the embedding liquid；
(3) embedding drop is added in crosslinked fluid, 4 DEG C stand for 24 hours, and the solid particle that washing, collection obtain is to be used for
It degrades the microbial inoculum of triazine herbicide, the wet thallus content of bacillus albus (Leucobacter sp.) JW-1 is in the microbial inoculum
30g/L, polyvinyl alcohol content 120g/L, sodium alginate content be 20g/L.
The technical solution further limited is as follows：
The specific preparation manipulation of wet thallus of bacillus albus described in step (2) (Leucobacter sp.) JW-1 is as follows：
Aseptically, bacillus albus (Leucobacter sp.) JW-1 is inoculated in solid medium, is placed in constant incubator
It is inside protected from light culture 4 days, 30 DEG C ± 1 DEG C of cultivation temperature, obtains activated spawn；Strain after activation is inoculated in 100mL enrichment training
In nutrient solution, 30 DEG C ± 1 DEG C, 150r/min constant-temperature table is interior to cultivate 40h, obtains bacteria suspension；Bacteria suspension is centrifuged in 6000r/min
10min discards supernatant liquid, retains sediment；Precipitating is washed 3 times with 0.9% sodium chloride solution, obtains bacillus albus
The wet thallus of (Leucobacter sp.) JW-1.
The solid medium is beef extract-peptone solid medium：Beef extract 5g, peptone 10g, sodium chloride 10g,
Agar 18g, distilled water 1L, pH are adjusted to 7.5,101KPa, 121 DEG C of sterilizing 20min.
The enrichment culture liquid is beef extract-peptone fluid nutrient medium：Beef extract 5g, peptone 10g, sodium chloride 10g,
Distilled water 1L, pH are adjusted to 7.5,101KPa, 121 DEG C of sterilizing 20min.
Crosslinked fluid described in step (3) be added to the water by boric acid and calcium chloride it is uniformly mixed, in pressure 101KPa, temperature
Sterilize 20min under the conditions of 121 DEG C, and the cooling solution being configured to of room temperature, the content of boric acid is 50g/L, and the content of calcium chloride is
20g/L。
Bacillus albus (Leucobacter sp.) JW-1 has good degradation effect to a variety of triazine herbicides, can be fast
Degradation (- SCH containing methyl mercapto of speed3) and chloro (- Cl) triazine herbicide.Free bacillus albus easily flows in the environment
Lose, adaptive capacity to environment it is poor, therefore the bacterium bag is buried and fixed microbial inoculum is made applies to the biological prosthetic of triazine herbicide
Technical field has biggish biological prosthetic meaning and economic benefit.
Advantageous effects of the invention embody in the following areas：
(1) microbial inoculum of the invention, the material used by it (polyvinyl alcohol, sodium alginate etc.) is harmless to microorganism, can be with
The preferable degrading activity for retaining bacillus albus (Leucobacter sp.) JW-1.Bacillus albus (Leucobacter sp.)
The thallus of JW-1 is embedded in microbial inoculum, can to avoid water body flowing and cause the loss of thallus, reduce mechanical friction pair
It is injured caused by thallus.Production cost is low, and direct plungeing into water body or soil can be used, and has broad application prospects.
(2) microbial inoculum of the invention can quickly degrade the prometryn of residue in water, and the degradation of 100mg/L prometryn partly declines
Phase is only 1.1h；Degradation rate is more than 60% in 100mg/L prometryn 60 days in the soil.
(3) it can be used repeatedly four times for microbial inoculum of the invention, stills remain in the degradation rate of prometryn (100mg/L)
40% or more.
(4) microbial inoculum of the invention can degrade a variety of triazine herbicides, such as：Prometryn, symetryne, ametryn, green bristlegrass are gone
Saliva, Simanex, propazine and Garagard can be widely used in the residual contamination for repairing triazine herbicide.
Detailed description of the invention
Fig. 1 is the physical form figure of microbial inoculum of the present invention；
Fig. 2 is microbial inoculum of the present invention to prometryn degradation effect figure；
Fig. 3 is that microbial inoculum of the present invention recycles number and degradation effect figure to what prometryn was degraded；
Fig. 4 is degradation effect figure of the microbial inoculum of the present invention to prometryn in soil.
Specific embodiment
With reference to the accompanying drawing, the present invention is further described by embodiment；But embodiments of the present invention are not
It is limited to this.Bacillus albus used in following embodiment (Leucobacter sp.) JW-1 is deposited in Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number are CGMCC No：
11754, preservation date：On November 27th, 2015.
Embodiment 1
It prepares as follows for the operating procedure of the microbial inoculum for triazine herbicide of degrading：
(1) bacillus albus (Leucobacter sp.) JW-1 wet thallus is prepared
Aseptically, solid is inoculated in connecing collarium and dip a small amount of bacillus albus (Leucobacter sp.) JW-1
In culture medium, it is placed in constant incubator and is protected from light culture 4 days, 30 DEG C ± 1 DEG C of cultivation temperature, obtain activated spawn；After activating
Strain be inoculated with two rings in equipped with 100mL liquid enriched medium 250mL triangular flask in, 30 DEG C ± 1 DEG C, 150r/min constant temperature
40h is cultivated in shaking table, obtains bacteria suspension；Bacteria suspension is centrifuged 10min in 6000r/min, liquid is discarded supernatant, retains precipitating；With
0.9% sodium chloride solution washing precipitating 3 times, obtains the wet thallus of bacillus albus (Leucobacter sp.) JW-1.
Wherein solid medium is beef extract-peptone solid medium, and formula is：Beef extract 5g, peptone 10g, chlorination
Sodium 10g, agar 18g, distilled water 1L, pH are adjusted to 7.5,101KPa, 121 DEG C of sterilizing 20min；Liquid enriched medium is beef extract
Peptone fluid nutrient medium, formula are：Beef extract 5g, peptone 10g, sodium chloride 10g, distilled water 1L, pH are adjusted to 7.5,
101KPa, 121 DEG C of sterilizing 20min.
(2) thallus embedding liquid is prepared
12g polyvinyl alcohol and 2g sodium alginate are sequentially added in 100mL water, stirred and evenly mixed, in high-pressure steam sterilizing pan
Interior 121 DEG C of sterilizings 20min, room temperature is cooling, obtains mixed liquor；
The wet thallus of 3g bacillus albus (Leucobacter sp.) JW-1 is added in 100mL mixed liquor, stirs and evenly mixs,
Obtain embedding liquid.
(3) preparation of crosslinked fluid
By 5g boric acid and with 2g calcium chloride be added 100mL water in dissolve, in high-pressure steam sterilizing pan 121 DEG C sterilizing
20min, room temperature cooling obtain crosslinked fluid.
(4) crosslinking prepares triazine herbicide degradation bacterium preparation
250mL triangular flask equipped with 100mL crosslinked fluid is placed on magnetic stirring apparatus, 100mL is embedded into liquid syringe
Slowly instill in crosslinked fluid, it is fixed under the conditions of 4 DEG C to collect microbial inoculum for 24 hours, 3 times are washed with 0.9% sterile sodium chloride solution, i.e.,
Obtain the microbial inoculum of degradation triazine herbicide；The content of the wet thallus of bacillus albus (Leucobacter sp.) JW-1 in microbial inoculum
Content for 30g/L, polyvinyl alcohol is 120g/L, the content of sodium alginate is 20g/L.
As seen from Figure 1, microbial inoculum is round granular, and surface is smooth, and elasticity is good, and mechanical strength is good, and transparent performance is good.
Embodiment 2
Degradation experiment of the microbial inoculum of degradation triazine herbicide to prometryn
Degradation experiment of the microbial inoculum of degradation triazine herbicide to prometryn in 1 water body
(1) prepared by minimal medium (Mineral Salt Medium, hereinafter referred to as MSM)：
MgSO4·7H2O, 0.4 gram；FeSO4·7H2O, 0.002 gram；K2HPO4, 0.2 gram；(NH4)2SO4, 0.2 gram；
CaSO4, 0.08 gram；Distilled water 1L；PH is adjusted to 7.5, and prepared MSM is spare through 121 DEG C of high pressure sterilization 20min.
(2) into the MSM of the 20mL containing 100.0mg/L prometryn, the triazine herbicide degradation bacteria prepared is added
Microbial inoculum 0.3g, cultivate 0 on 37 DEG C, 150r/min shaking table, 1,2,3,4,6,8,10, after 12h, it is dense to extract detection prometryn remaining
Degree.The microbial inoculum of the present invention of wet thallus is not added as blank control in setting simultaneously, and control and processing are all provided with 3 repetitions.
Experimental result is as shown in Figure 2, and microbial inoculum of the present invention is 1.1h to the degradation half life of prometryn, and the degradation rate after 6h reaches
To 96.72% or more, basic degradation is complete.
(3) microbial inoculum of triazine herbicide of degrading recycles experiment：To the 20mL's containing 100.0mg/L prometryn
In MSM, the microbial inoculum 0.3g of the degradation triazine herbicide prepared is added, after cultivating 12h on 37 DEG C, 150r/min shaking table,
Used microbial inoculum is filtered out, with MSM washing 3 times, reuses, detects its degradation effect, operating procedure is same as above.
Experimental result is as shown in Figure 3, and microbial inoculum of the present invention is being recycled 2 times, is still able to maintain to the degradation rate of prometryn
90.0% or more；Microbial inoculum is being recycled 3 times, is still able to maintain 60.0% or more to the degradation rate of prometryn.
Microbial inoculum of the present invention is directly added into sewage in practical application, 15g microbial inoculum is added in every 1L sewage.Bacterium of the present invention
Agent can be recycled 3 times, remain to reach 60.0% or more degradation effect.
Degradation experiment of the microbial inoculum of 2 degraded in soil triazine herbicides to prometryn
The soil of the 100g (dry weight) containing 100.0mg/L prometryn is added into the triangular flask of 250mL, and is added simultaneously
The microbial inoculum 10g of degradation triazine herbicide, after mixing, is cultivated in 37 DEG C of constant incubators, and each 3 days into soil plus water makes
Its moisture content is maintained between 25%-30% and shakes up the soil in bottle, after 0,6,12,18,24,30,45,60d, soil sampling
Detect prometryn remaining concentration in soil.The microbial inoculum of the present invention of wet thallus is not added as blank control, control and place in setting simultaneously
Reason is all provided with 3 repetitions.
Experimental result is as shown in Figure 4, and 12d microbial inoculum of the present invention reaches 44.4%, 60d's to the degradation rate of prometryn in soil
Degradation rate is 63.1%.
Embodiment 3
Degradation experiment of the microbial inoculum of degradation triazine herbicide to other triazine herbicides
Respectively to containing 50.0mg/L ametryn, symetryne, atrazine, propazine, Simanex 20mL MSM in, add
The microbial inoculum 0.5g for entering triazine herbicide of degrading after cultivating 2,4d respectively on 37 DEG C, 150r/min shaking table, extracts detection triazine
Class herbicide remaining concentration.The microbial inoculum of the present invention of wet thallus is not added as blank control in setting simultaneously, and control and processing are all provided with 3
It repeats.
Table 1
It the results are shown in Table 1, the microbial inoculum for triazine herbicide of degrading is to atrazine, propazine, Garagard, Simanex, ametryn
There is good degradation effect with symetryne, after 2d, atrazine, propazine, Garagard, ametryn and symetryne almost all drop
Completely, the degradation rate of Simanex is close to 80% for solution；After 4d, the degradation rate of Simanex reaches 90%.Therefore microbial inoculum of the present invention is to dirt
It is remaining to contaminate six kinds of triazine herbicides such as atrazine, propazine, Garagard, Simanex, ametryn and the symetryne in environment
Degradation has important reparation meaning.
Claims (6)
1. a kind of microbial inoculum for triazine herbicide of degrading, it is characterised in that：The microbial inoculum is with sodium alginate, polyvinyl alcohol
Cross-linking products are carrier, and embedding bacillus albus JW-1 is made；
Bacillus albus in the microbial inoculum（Leucobactersp.）The wet thallus content of JW-1 is 30 g/L, polyvinyl alcohol content
Content for 120 g/L, sodium alginate is 20 g/L；
Microbial inoculum is round granular, and surface is smooth, and elasticity is good, and mechanical strength is good, and transparent performance is good；
The bacillus albus（Leucobactersp.）JW-1 bacterium is deposited in China General Microbiological culture presevation administrative center,
Deposit number is CGMCC No：11754, preservation date：On November 27th, 2015.
2. the method described in preparation claim 1 for the microbial inoculum for triazine herbicide of degrading, it is characterised in that：Operating procedure is such as
Under：
（1）Polyvinyl alcohol and sodium alginate are sequentially added in water, stirred and evenly mixed, in 121 DEG C of 101 KPa of pressure, temperature conditions
20 min of lower sterilizing, room temperature is cooling, obtains mixed liquor；The content of polyvinyl alcohol is 120 g/L, sodium alginate in the mixed liquor
For 20 g/L；
（2）Bacillus albus is added in mixed liquor（Leucobactersp.）The wet thallus of JW-1, stirs and evenly mixs, and obtains thallus
Embed liquid；Bacillus albus in the embedding liquid（Leucobactersp.）The content of the wet thallus of JW-1 is 30 g/L；
（3）Embedding drop is added in crosslinked fluid, 4 DEG C of 24 h of standing, the solid particle that washing, collection obtain is to be used to drop
Solve the microbial inoculum of triazine herbicide, bacillus albus in the microbial inoculum（Leucobactersp.）The wet thallus content of JW-1 is 30
G/L, polyvinyl alcohol content are 120 g/L, the content of sodium alginate is 20 g/L；The crosslinked fluid by 5 g boric acid and with 2 g chlorine
Change dissolution in calcium 100 mL water of addition to be made.
3. preparation method according to claim 2, it is characterised in that step（2）Described in bacillus albus
（Leucobactersp.）The specific preparation manipulation of the wet thallus of JW-1 is as follows：
Aseptically, by bacillus albus（Leucobactersp.）JW-1 is inoculated in solid medium, is placed in constant temperature training
It supports and is protected from light culture 4 days in case, 30 DEG C ± 1 DEG C of cultivation temperature, obtain activated spawn；Strain after activation is inoculated in 100 mL
In enrichment culture liquid, 30 DEG C ± 1 DEG C, 150 r/min constant-temperature tables are interior to cultivate 40 h, obtains bacteria suspension；By bacteria suspension in
6000 r/min are centrifuged 10 min, discard supernatant liquid, retain sediment；Precipitating is washed 3 times with the sodium chloride solution of 0.9 %, is obtained
To bacillus albus（Leucobactersp.）The wet thallus of JW-1.
4. preparation method according to claim 3, it is characterised in that：The solid medium is beef extract-peptone solid
Culture medium.
5. preparation method according to claim 3, it is characterised in that：The enrichment culture liquid is beef extract-peptone liquid
Culture medium.
6. preparation method according to claim 2, it is characterised in that：Step（3）Described in crosslinked fluid by boric acid and chlorine
Change calcium and be added to the water uniformly mixed, sterilize 20 min under the conditions of 101 KPa of pressure, 121 DEG C of temperature, and room temperature cooling is configured to
Solution, the content of boric acid is 50 g/L, and the content of calcium chloride is 20 g/L.
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| CN107601681B (en) * | 2017-11-02 | 2020-11-03 | 杭州富阳优信科技有限公司 | Composite microbial inoculum for repairing metamitron polluted water body |
| CN107585977B (en) * | 2017-11-06 | 2020-09-25 | 杭州富阳优信科技有限公司 | Method for repairing metamitron polluted water body by biological method |
| CN108410748B (en) * | 2017-12-07 | 2021-08-24 | 安徽农业大学 | Microbial inoculum for degrading triazine herbicides and preparation method thereof |
| CN108503039A (en) * | 2018-03-15 | 2018-09-07 | 湖南大学 | A method of utilizing Penicillium notatum magnetic nanometer composite material degradation Atrazine |
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| US4745064A (en) * | 1983-11-04 | 1988-05-17 | Ciba-Geigy Corporation | Process for the degradation of s-triazine derivatives in aqueous solutions |
| CN1257265C (en) * | 2004-06-07 | 2006-05-24 | 南京农业大学 | Triazine herbicide residual degrading strain and strain therefrom |
| CL2007001982A1 (en) * | 2007-07-06 | 2008-01-04 | Univ Tecnica Federico Santa Maria Utfsm | Pseudomonas sp strain capable of degrading or mineralizing s-triazines corresponding to the pseudomonas sp. mph41 with deposit number nrrl b-30908, capable of degrading or mineralizing s-triazines in the presence of urea, nitrate or ammonium; bioremediation product comprising said bacterium and a culture medium; method to bioremediate environment contaminated with s-triazines which comprises applying the aforementioned product |
| CN105112317A (en) * | 2015-06-17 | 2015-12-02 | 哈尔滨工业大学宜兴环保研究院 | High-efficiency atrazine degrading bacterium and screening method thereof, and application of high-efficiency atrazine degrading bacterium in biodegradation of atrazine in farmland waste water |
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